We are designing peptides with novel staples incorporating a variety of chemical moieties, such as aromatic and heteroaromatic rings, H-bond donors and acceptors, alkynes, alkenes, etc. These designs are first evaluated by computational chemistry for the induction of alpha-helicity and for physical properties (logP, PSA, etc.). Additionally, we are developing a novel synthetic approach, in which we first construct the staple as a bis-amino acid synthon (two amino acids linked through a staple) that is then used in automated peptide synthesis. Our approach differs from the current literature, in which staples are made after the peptide has been synthesized. This current approach limits the chemical diversity of the staples, whereas our novel approach should open up greater synthetic chemistry options for making staples (e.g., Suzuki reaction, reductive amination, etc.), increasing the diversity of chemical moieties that could be incorporated. We have optimized all the synthetic steps for our new approach. Adaptation of this approach to automatic peptide synthesis is near completion. Once that is achieved, we will have a high-throughput synthesis available to quickly generate a variety of stapled peptides for further evaluation. This project represents the first use of automated peptide synthesis technology at NCATS, which is now available to all groups in the intramural program.